Antimicrobial compositions

ABSTRACT

Disclosed herein are 2-aminoalkyl-3-aryl-hetero-indenes useful as antimicrobial agents.

This is a division of application Ser. No. 636,517 filed Dec. 1, 1975,now U.S. Pat. No. 3,996,159, which in turn is a division of applicationSer. No. 473,625 filed May 28, 1974, now U.S. Pat. No. 3,944,672 whichin turn is a continuation-in-part of Ser. No. 314,899 filed Dec. 13,1972, now abandoned.

This invention is also continuation-in-part of application Ser. No.314,899 filed Dec. 13, 1972.

This invention relates to antimicrobial compounds and compositionscontaining them, to processes for their preparation, and to their use asantimicrobial agents. The compounds may be used in foods and in medicineand in industrial fields as antimicrobial agents. "Antimicrobial" asused herein refers to antibacterial, antifungal or antiprotozoalactivity.

The antimicrobial compounds of the invention are2-aminoalkyl-3-aryl-hetero-indenes of the formula: ##STR1## wherein n is0 or 1: R₂ is hydrogen or amino lower alkylene having 1 to 3 carbonatoms; R₁ is carbonyl or lower alkylene having 1 to 3 carbon atoms withthe provisio that when R₂ is hydrogen, R₁ is lower alkylene; T is NH, Sor O; X is halogen, nitro, or trifluoromethyl; Y is hydrogen, halogen,methyl or trifluoromethyl; M is hydrogen, halogen or methyl with theprovisio that when X is nitro, M is halogen; Z is hydrogen, halogen,nitro or methyl; and the acid addition salts thereof.

"Heteroindene", as used herein, is an indene wherein the carbon atom inthe 1-position is replaced by a hetero atom selected from N_(H), S, orO, i.e. indole, benzothiophene and benzofuran, respectively.

As used herein the term "lower alkylene" refers to methylene, ethylene,propylene, and isopropylene and cyclopropylene. The term "halogen" asused herein comprehends fluorine, chlorine, bromine and iodine. Chlorois the preferred X substituent. A preferred location of the indole ringfor the X moiety is the 5-position. Preferaby M is ortho-halo, withortho-fluoro or ortho-chloro being especially preferred. T is preferablyNH although it may be S or O. The term "amino lower alkylene" refers toradicals represented by the formula --R₃ --NH₂ wherein R₃ is loweralkylene as defined above. "n" preferably is 0.

A particularly useful group of anti-microbial2-aminoalkyl-3-phenyl-hetero-indenes within formula I has the formula:##STR2## wherein n is 0 or 1, X is halogen or trifluoromethyl, M ishydrogen, bromo or chloro, and one of Y and Z is 2-fluoro or 2-chloroand the other is hydrogen, halo, nitro or trifluoromethyl. It isparticularly desireable for the M,Z-substituted phenyl group to be2-fluorophenyl, 2,6-difluorophenyl, or 2,4-dichlorophenyl.

Particularly preferred compounds within this group include:

2-aminomethyl-5-chloro-3-(2-fluorophenyl)indole,

2-aminomethyl-5-chloro-3-(2,6-difluorophenyl)indole,

2-aminomethyl-5-chloro-3-(2,6-dichlorophenyl)indole,

2-aminomethyl-5-chloro-3-(2,4-dichlorophenyl)indole,

2-aminomethyl-5,7-dichloro-3-(2,4-dichlorophenyl)indole,

2-aminomethyl-5,6-dichloro-3-(2,4-dichlorophenyl)indole,

2-aminomethyl-6-chloro-3-(2,4-dichlorophenyl)indole,

2-aminomethyl-5-chloro-6-bromo-3-phenylindole, and

5,6-dichloro-3-(2,4-dichlorophenyl)indole-2-(N-β-aminoethyl)carboxamide.

Other particularly interesting compounds within formula I include:

2-(1-aminoethyl)-5-chloro-3-phenylindole,

2-(1-aminoethyl)-5-chloro-6-bromo-3-phenylindole,

2-aminomethyl-5-chloro-3-(benzyl)indole,

2-aminomethyl-5-chloro-3-(3,4-dichlorophenyl)indole, and

2-[N-(β-aminoethyl)aminoethyl]-5-chloro-3-phenylindole hydrochloride.

The acid addition salts can sometimes be used more conveniently than theindenes themselves; for example, because the salts have more convenientphysical properties such as crystalline form or solubility, or becausethe salts are more readily purified by recrystallization, than theindenes. When the salts are used in food or medicine, the anion must, ofcourse, be substantially non-toxic at the concentration or dosage used;when the salts are used in technical fields such as the preservation ofpaper, leather, or photographic goods, the anion need not necessarily benon-toxic.

Although some of the compounds of formula II have been proposed asintermediates in the preparation of benzodiazepines, many are novel; inparticular, compounds in which M is halogen and/or Y and Z are nothydrogen are novel. Novel compounds within formula I include:

2-aminomethyl-5-chloro-3-(2,6-difluorophenyl)indole,

2-aminomethyl-5-chloro-6-bromo-3-phenylindole,

2-aminomethyl-5-chloro-3-(2,4-dichlorophenyl)indole,

2-(1-aminoethyl)-5-chloro-6-bromo-3-phenylindole,

2-aminomethyl-5,7-dichloro-3-(2,4-dichlorophenyl)indole,

2-aminomethyl-5,6-dichloro-3-(2,4-dichlorophenyl)indole,

2-aminomethyl-5-chloro-3-(3,4-dichlorophenyl)indole,

2-aminomethyl-5-chloro-3-(2,4-dichlorophenyl)-6-nitroindole,

2-aminomethyl-6-chloro-3-(2,4-dichlorophenyl)indole,

2-aminomethyl-5-iodo-3-(2,4-dichlorophenyl)indole,

2-aminomethyl-5-trifluoromethyl-3-(2,4-dichlorophenyl indole,

2-aminomethyl-6-chloro-3-(2,4-dichlorophenyl)indole,

2-aminomethyl-5-chloro-3-(3,4-dimethylphenyl)indole.

Still other compounds within the formula I are:

2-aminopropyl-7-iodo-3-(3-nitrophenyl)indole,

2-(2'-aminopropyl)-5-trifluoromethyl-3-(2-fluoro-4-nitrophenyl)indole,

2-aminomethyl-3-(2-nitrophenyl)indole,

2-aminopropyl-7-bromo-3-(2-trifluoromethylphenyl)indole.

The 2-aminoalkyl-3-phenylindenes of the formula II and their acidaddition salts can be prepared by standard methods, know for thepreparation of substituted indenes or of amines; for example, processesdisclosed in the following literature: U.S. Pat. Nos. 3,697,508;3,558,604 and 3,558,603; Belgian Pat. No. 724,993; and"Benzodiazepines", Inaba, Ishizumi and Yamamoto, Chem. Pharm. Bull.,19(2), pages 263-272 (1971).

The 3-phenylindole nucleus itself is normally most conveniently obtainedby a Fischer indole synthesis, or by an equivalent reaction in which abenzenediazonium salt reacts with an ester of a 2-benzylacetoacetic acidin the presence of alkali and the product is cyclised by heating in thepresence of acid or in ethylene glycol. Further modifications,especially transformation of the substituent at the 2-position, and/orremoval of a blocking or protecting group at the 1-position, may then beeffected.

The 2-aminoalkyl-3-phenylbenzo [b] thiophenes can be prepared frommethyl 3-phenylbenzo [b] thiophene-2-carboxylate, known from Krubsackand Higa in Tetrahedron Letters No. 47, page 4823 (1972), by standardmethods. The 2-aminoalkyl-3-phenylbenzo [b] furans can be prepared from2-cyano-5-chloro-3-phenylbenzofuran, known from standard methods.

Typical final steps are, reduction of a precursor compound of theformula: ##STR3## wherein Q is a group directly reducible to R₁ NHR₂.The reduction can be effected by catalytic hydrogenation, by lithiumaluminium hydride; electrolytically, etc.

A cyano group Q is most conveniently reduced with a boron hydride,especially borane, or with a complex metal hydride such as lithiumaluminium hydride, calcium borohydride, or sodium borohydride in thepresence of aluminium chloride or of boron trifluoride. The inertsolvent will normally be an ether solvent, e.g., diethyl ether,tetrahydrofuran or dioxan. Compounds in which Q is a cyano group mayalso be reduced electrolytically.

Other suitable Q groups are carbamoyl or thiocarbamoyl, nitro alkyl, anoxime, ether or ester thereof, a ketimine, a hydrazine, or asemicarbazone.

Indenes wherein R₂ is an amino lower alkylene may be prepared byreaction of the corresponding carboxylate with the appropriate amine.

The 2-aminoalkyl-3-phenyl-heteroindenes may be isolated from theforegoing processes as the free bases or as acid addition salts or maybe interconverted into acid addition salts e.g., hydrochlorate,sulfurate, nitrate, phosphate, acetate or succinate, phenolate and thelike. The acid may be so chosen as to additionally impart activity, e.g.hexylresoicinol, to enhance the antimicrobial spectrum.

The following examples illustrate the preparation of compounds of theformula I; Example 1 shows the preparation of a preferred compound bythe standard reduction of the corresponding nitrile with lithiumaluminium hydride, whereas Example 2 illustrates a slightly differentfinal step and also the preparation of the appropriate precursors.Examples 3 and 4 illustrates the most preferred compounds of theinvention. Examples 5 and 6 respectively illustrate a thiophene andfuran compound according to the invention, while Example 7 illustrates apreferred indole wherein R₂ is an amino lower alkylene.

EXAMPLE 1 2-Aminomethyl-5-chloro-3-(2-fluorophenyl)indole

Add a solution of 7.5 g. (0.0277 mol) of5-chloro-3-(2-fluorophenyl)indole-2-carbonitrile in 100 ml of dry etherto a stirred suspension of 1.25 g. (0.033 mol) of lithium aluminiumhydride in 200 ml of dry ether. Reflux the reaction mixture for 4 hours.Cool to room temperature and slowly add 3 ml of water to quench thereaction. Filter the mixture and dry overnight over magnesium sulfate.

Filter the solution, evaporate to dryness and recrystallize the residuefrom a mixture of methylene chloride and petroleum ether to give thetitle compound. The hexylresorcinol salt melts at 150°-152° C.

EXAMPLE 2 2-Aminomethyl-5-chloro-3-(2,6-difluorophenyl)indole Step A:N-[2-(2,6-difluorobenzoyl)-4-chloro]phenylglycinonitrile

A mixture of 5 g. (0.0186 mol) of2-amino-5-chloro-2',6'-difluorobenzophenone, 3.9 g. (0.06 mol) ofpotassium cyanide and 2.8 g. (0.093 mol) of paraformaldehyde is treatedwith 25 ml of glacial acetic acid saturated with dry hydrogen chloride.The mixture is refluxed for 15 minutes, then poured onto 800 ml of icewater. The resulting precipitate is filtered off, washed with water andrecrystallized from methanol (m.p. 175°-176°). Recrystallization affordsyellow needles; m.p. 182°-183°.

Step B: 5-chloro-3-(2,6-difluorophenyl)indole-2-carbonitrile

A solution of N-[2-(2,6-difluorobenzoyl)-4-chloro]phenylglycinonitrile(2.0g) in 20 ml of dry tetrahydrofuran is treated with a large excess (4ml) of trifluoroacetic anhydride. The mixture is refluxed for 4 hours,and solvent and excess of reagent are removed under vacuum. The residueis twice treated with dry tetrahydrofuran and each time evaporated todryness. Finally, the residue is again dissolved in dry tetrahydrofuranand treated with 1 g of sodium hydride (57% in mineral oil).

The mixture is refluxed for one hour, cooled to room temperature andpoured into a mixture of ice, water and chloroform. The organic layer isseparated, washed with water and dried over magnesium sulfate.Filtration and evaporation yield a semisolid, which is filtered throughsilica gel to give the title compound; m.p. 199°-200°.

Step C: 2-aminomethyl-5-chloro-3-(2,6-difluorophenyl)indole

Borane in tetrahydrofuran (7 ml of a 1M solution) is added to a solutionof 1 g (3.46 mmol) of 5-chloro-3-(2,6-difluorophenyl)indole-2-carbonitrile in 15 ml of dry tetrahydrofuran. The solution isstirred at room temperature for 2 hours and then refluxed for 1/2 andcooled to room temperature. 5 ml of 5N aqueous NCl is added dropwise andthe mixture is refluxed for 20 minutes. The mixture is then poured ontoa mixture of ice and aqueous 10 percent ammonia, and the precipitate isextracted with chloroform. The chloroform layer is washed with water anddried over magnesium sulfate, filtered and evaporated to dryness.Recrystallization from chloroform (charcoal) affords colorless crystals;m.p. 212°-214°.

EXAMPLE 3 2-Aminomethyl-6-bromo-5-chloro-3-phenylindole Step A: Ethyl6-bromo-5-chloro-3-phenylindole-2-carboxylate

Add 11 ml (33 g.) of bromine to a mixture of ethyl5-chloro-3-phenylindole-2-carboxylate (30 g.) in 100 ml of acetic acidat 10°-20°. Stir overnight and then add a solution of sodium bisulfite.Filter and wash the precipitate with water. Stir the solid with methanoland filter to yield the title compound. Recrystallization from methanolyields the analytical sample; m.p. 205°-206° C.

Step B: 6-Bromo-5-chloro-3-phenylindole-2-carboxylic acid

Reflux the ester (13 g.) of step A in 100 ml of ethanol with 150 ml of10% sodium hydroxide solution for 2 hours. Evaporate off most of themethanol and, after cooling the mixture, add 5% hydrochloric acid.Collect the precipitate and dry to obtain the title compound; m.p.241°-242° C.

Step C: 6-Bromo-5-chloro-3-phenylindole-2-carboxamide

Mix the acid (6 g.) of step B with 20 ml of thionyl chloride and boilunder reflux for 0.5 hour. Evaporate the solution to dryness. Addtetrahydrofuran and evaporate twice to remove any remaining thionylchloride. Add tetrahydrofuran (80 ml) and bubble ammonia gas through thesolution. Add water to dissolve the precipitate at which time an oilseparates. Add methanol to the oil to yield crystals which are collectedand dried to yield the title compound.

Step D: 6-Bromo-5-chloro-3-phenylindole-2-carbonitrile

Reflux the amide (4.5 g.) of step C with phosphorous oxychloride (20 ml)for 2 hours. Cautiously pour the mixture, while still hot, ontoice-water. Collect the precipitate and dry to yield the title compound.

Step E: 2-Aminomethyl-6-bromo-5-chloro-3-phenylindole

Reflux the nitrile (10 g.) of step D in 100 ml of 1 molar borane intetrahydrofuran for 4 hours, cool and then cautiously add 7 ml ofconcentrated hydrochloric acid. Evaporate the solution and add 5% sodiumhydroxide with stirring. Collect the solid, dissolve in ethanol andprecipitate with water. Collect and dry the precipitate to yield thetitle compound. Recrystallize from benzene; m.p. 162°-163° C. Thehydrochloride melts at 202°-204° C.

EXAMPLE 4 2-Aminomethyl-5-chloro-3-(2,4-dichlorophenyl)indole Step A:N-[2-(2,4-dichlorobenzoyl)-4-chloro]phenylglycinonitrile

Mix together 2-amino-2',4',5-trichlorobenzophenone (5 g.), potassiumcyanide (3.25 g.), and paraformaldehyde (2.5 g.). Then add 12.5 ml ofacetic acid saturated with hydrogen chloride and 12.5 ml of acetic acid.Keep the temperature below 70° for several minutes and then boil underreflux for 5 minutes. Cool and pour onto ice-water. Collect theprecipitate and extract with chloroform. Treat this solution with sodiumcarbonate solution and then water and finally dry (MgSO₄). Evaporate toyield the title compound. Recrystallize from methylenechloride-petroleum ether; m.p. 166°-167°.

Step B: 5-Chloro-3-(2,4-dichlorophenyl)indole-2-carbonitrile

Dissolve the glycinonitrile of step A (40 g.) in 175 ml of drytetrahydrofuran. Add 50 ml of trifluoroacetic anhydride. Boil underreflux overnight. Remove the solvent under nitrogen. Add more solventand remove twice. Dissolve the crystalline residue in tetrahydrofuran,and treat with 20 g. of 57% sodium hydride in mineral oil. Boil underreflux for 30 minutes and pour onto icewater. Stir and collect thesolid. Wash with water, dry and wash with petroleium ether. Crystallizefrom methylene chloride-petroleum ether to obtain the title compound;m.p. 181°-182°.

Step C: 2-Aminomethyl-5-chloro-3-(2,4-dichlorophenyl)indole

Dissolve the nitrile (22 g.) of step B in a mixture of 137 ml of 1 molarborane in tetrahydrofuran and 100 ml of tetrahydrofuran and stir for 4hours. Boil under reflux for 0.5 hour, cool and add 5 N hydrochloricacid. Boil under reflux for 1 hour and pour onto a mixture of ice and10% ammonia. Extract with chloroform, wash and dry (MgSO₄). Evaporate toyield the title compound. Crystallize from ether-petroleum ether toobtain the analytical sample; m.p. 179°-180°. To prepare thehydrochloride salt, dissolve this material in ethanol, add concentratedhydrochloric acid, collect and dry the material; m.p. 226°-228°.

EXAMPLE 5 2-Aminoethyl-5-bromo-3-phenylbenzo[b]thiophene hydrochlorideStep A: Methyl 5-bromo-3-phenylbenzo[b]thiophene-2-carboxylate

Add 29.3 g. bromine (10 ml; 0.37 mole) to 8.0 g. (0.03 mole) of methyl3-phenylbenzo[b]thiophene-2-carboxylate in 100 ml of acetic acid andstir the mixture overnight at room temperature. Add a saturated solutionof sodium bisulfite. Decant the supernatant and wash the oil with water.Triturate with methanol. Recrystallize from methanol to yield the titlecompound; m.p. 125°-126°.

Step B: 5-Bromo-3-phenylbenzo[b]thiophene-2-carboxylic acid

Heat three grams of methyl5-bromo-3-phenylbenzo[b]thiophene-2-carboxylate in a mixture of 50 ml ofmethanol and 30 ml of 10% sodium hydroxide, under reflux for 1 hour. Addconcentrated hydrochloric acid to yield the title compound as aprecipitate. Recrystallize from benzene; m.p. 265°-266°.

Step C: 5-Bromo-3-phenylbenzo[b]thiophene-2-carboxamide

Reflux 2.8 g. 5-bromo-3-phenylbenzo[b]thiophene-2-carboxylic acid(0.0084 mole) and 10 ml of thionyl chloride in 15 ml of drytetrahydrofuran for one hour, evaporate to dryness, add 50 ml oftetrahydrofuran twice and evaporated each time to dryness. Dissolve acidin 70 ml of tetrahydrofuran, cool to about 10° and add with stirring 10ml of concentrated ammonia. The title compound is obtained as a whitesolid; m.p. 201°-202°.

Step D: 5-Bromo-2-cyano-3-phenylbenzo[b]thiophene

Reflux 2.1 g. 5-bromo-3-phenylbenzo[b]thiophene-2-carboxamide (0.0063mole) together with 10 ml. of phosphorus oxychloride for 11/2 hours andthen carefully pour onto ice water. Filter off the white precipitate.Dry. Recrystallize the title compound from methanol; m.p. 145°-146°.

Step E: 2-Aminomethyl-5-bromo-3-phenylbenzo[b]thiophene hydrochloride

Treat 1.4 g. 5-bromo-2-cyano-3-phenylbenzo[b]thiophene (0.0045 mole) in40 ml of tetrahydrofuran, with 10 ml of 1 molar borane intetrahydrofuran. Reflux the solution for 11/2 hours. Slowly add 10 ml of5% hydrochloric acid. Heat the mixture for 1/2 hour, cool and addsufficient sodium hydroxide solution to basicity. Extract the solutionwith ether and wash twice with water. Dry the ether layer (MgSO₄),filter and mix with 50 ml of ether saturated with hydrogen chloride. Drythe precipitate to yield the title compound; m.p. over 225° (decomp).

EXAMPLE 6 2-Aminomethyl-5-chloro-3-phenylbenzofuran hydrochloride

Treat 2.0 g. 2-cyano-5-chloro-3-phenylbenzofuran (0.008 mole) in 30 mlof tetrahydrofuran with 18 ml of borane in tetrahydrofuran (1 msolution). Reflux for 2 hours. Cool, add 10 ml of 10% hydrochloric acidand reflux for 1/2 hour. Evaporate the solution to dryness. Dissolve theresidue in 50 ml of ethanol. Add 30 ml of ethanol saturated withhydrogen chloride. Dry the precipitate obtained to yield the titlecompound; m.p. 254°-256°.

EXAMPLE 75,6-dichloro-3-(2,4-dichlorophenyl)indole-2-N-(β-aminoethyl)carboxamideStep A: Ethyl 5,6-dichloro-3-(2,4-dichlorophenyl)indole-2-carboxylate

Prepare a solution of 3,4-dichlorophenyl-diazonium chloride by treating8.2 g. of 3,4-dichloroaniline in 25 ml of concentrated hydrochloric acidwith a solution of 3.5 g. of sodium nitrite in 35 ml of water at -5°.Dissolve ethyl 2-(2,4-dichlorobenzyl) acetoacetate (14.3 g.) in 100 mlof ethanol with 8.3 g. of potassium hydroxide in 8.3 ml of water andcool to -5°. Add the solution of the diazonium salt to the lattersolution, keeping the temperature at 0°, and stir for 15 minutes.Extract with ether and wash the ether solution with two 75 ml portionsof 5% sodium hydroxide and then with water and dry (MgSO₄). Evaporate toyield the intermediate: ethyl 2,4-dichlorophenylpyruvate3,4-dichlorophenyl hydrazone. The analytical sample is obtained fromethanol; m.p. 160°-161°. Dissolve the hydrazone (125 g.) in 700 ml of20% ethanolic sulfuric acid and boil under reflux for 2 hours. Cool toroom temperature and collect the crystalline product. Wash with 50%aqueous alcohol. Recrystallize from ethanol; m.p. 230°-231°.

Step B:5,6-dichloro-3-(2,4-dichlorophenyl)indole-2-N-(β-aminoethyl)carboxamide

Boil a mixture of 1.0 g. of ethyl5,6-dichloro-3-(2,4-dichlorophenyl)indole-2-carboxylate, 1 ml ofethylene diamine and 5 ml of dry toluene under reflux for 24 hours. Thencool the mixture to room temperature, dilute with benzene, wash withwater and dry over MgSO₄. Filter off the MgSO₄ and evaporate off thesolvent to yield a solid residue, which upon crystallization from CHCl₃-petroleum ether gives the title compound; 600 mg; m.p. 216°-218°.

The following compounds may be prepared in a final step similar to thatof Examples 1-7:

2-(1-aminoethyl)-6-bromo-5-chloro-3-phenylindole,

2-aminomethyl-5,7-dichloro-3-(2,4-dichlorophenyl)indole,

2-aminomethyl-5,6-dichloro-3-(2,4-dichlorophenyl)indole,

2-aminomethyl-5-chloro-3-(3,4-dichlorophenyl)indole,

2-aminomethyl-5-chloro-3-(2,4-dichlorophenyl)-6-nitroindole,

2-aminomethyl-6-chloro-3-(2,4-dichlorophenyl)-5-fluoroindole,

2-aminomethyl-3-(2,4-dichlorophenyl)-5-iodoindole,

2-aminomethyl-3-(2,4-dichlorophenyl)-5-trifluoromethylindole,

2-aminomethyl-6-chloro-3-(2,4-dichlorophenyl)indole,

2-aminomethyl-5-chloro-3-(2,4-dichlorophenyl)-6-methoxyindole,

2-aminomethyl-5-chloro-3-(2-fluorophenyl)benzo[b]thiophene,

2-aminomethyl-5,6-dichloro-3-(2-fluorobenzyl)benzo[b]thiophene,

2-aminomethyl-6-bromo-3-(2,6-dichlorobenzyl)benzofuran,

2-aminomethyl-3-(2-chloro-6-methyl-phenyl)indole,

2-aminomethyl-5-chloro-3-(3,4-dimethylphenyl)indole,

2-aminomethyl-5,6-dichloro-3-(2,4 dichlorophenyl)benzo[b]thiophene,

2-aminoethyl-5-iodo-3-(2,4 dichlorophenyl)benzo[b]thiophene,

2-aminomethyl-3-(2-chloro-6 methyl-benzyl)benzo[b]thiophene,

2-aminomethyl-5-chloro-3-(2,4 dimethylphenyl)benzofuran,

2-aminomethyl-5-chloro-3-(2-chloro-6-trifluoromethylbenzyl)benzofuran,

2-aminomethyl-5-chloro-7-iodo-3-phenylbenzofuran hydrochloride,

5-chloro-3-(2-fluorophenyl)indole-2-N-(β-aminoethyl)carboxamide,

5,6-dichloro-3-(2,6-difluorophenyl)indole-2-N-(β-aminoethyl)carboxamide,

5,6-dichloro-3-(2,4-dichlorobenzyl)indole-2-N-(β-aminoethyl)carboxamide,and

2-[N-(β-aminoethyl)aminoethyl]-5-nitro-3-(2,4-di-trifluoromethylphenyl)indole.

The compounds of formula I and their non-toxic acid addition salts canbe used to treat diverse types of susceptible microbial infections.Furthermore, they are capable of preserving a wide variety ofpreparations including medical, veterinary, cosmetic and foodpreparations from microbial contamination: a stabilizing amount of sucha compound is incorporated in the preparation in which the preservationis desired.

Susceptibility can be readily determined by standard in vivo and invitro tests well known to the microbiologist. Genera of susceptiblemicroorganisms include bacteria, fungi, and protozoa.

Exemplifying susceptible bacterial microorganisms are Staphylococcusaureus, Streptococcus pyogenes C., Bacillus subtilis, Escherichia coliand Pseudomonas aeruginosa. Susceptible fungi include Candida albicans,Trichophyton mentagrophytes and Saccharomyces cerevisiae. Susceptibleprotozoal pathogens include Trychomonas vaginalis and Entamoebahistolytica.

Tables I to VII give the results of various in vitro and in vivo testsof representative 2-aminoalkyl-3-phenylindenes of formula II against avariety of microorganisms including bacteria, fungi and protozoa, andalso report the toxicity of these 2-aminoalkyl-3-phenylindenes. Thecompounds of the tests are as follows:

    ______________________________________                                        Compound    Name                                                              ______________________________________                                        A           2-aminomethyl-5-chloro-3-(2-fluoro-                                           phenyl)indole                                                     B           2-aminomethyl-5-chloro-3-(2,6-di-                                             fluoro-phenyl)indole                                              C           2-(1-aminomethyl)-5-chloro-3-phenyl-                                          indole                                                            D           2-aminomethyl-6-bromo-5-chloro-3-                                             phenylindole                                                      E           2-aminomethyl-5-chloro-5-(2,4-di-                                             chlorophenyl)indole                                               ______________________________________                                    

All the tests were carried out and (when necessary) scored understandard conditions. "MIC" means "minimum inhibitory concentration."

                  TABLE I                                                         ______________________________________                                        In vitro Antibacterial Activity of 2-aminoalkyl-3-phenyl-                     indoles of the formula I. Dilution in Mueller Hinton Agar pH 7.4              MIC (mcg/ml)                                                                              Compound                                                          Organism      A      B       C     D     E                                    ______________________________________                                         Staphylococcus aureus                                                         209P         7.5    7.5     7.5   3.0   3.0                                   Wood         3.0    7.5     7.5   3.0   3.0                                   Zeigler      3.0    7.5     3.0   3.0   0.8                                   Gray         7.5    7.5     7.5   3.0   3.0                                   59N          3.0    7.5     7.5   3.0   3.0                                  Streptococcus pyogenes                                                         C            7.5    17.5    7.5   3.0   3.0                                   27           7.5    7.5     7.5   3.0   3.0                                   Cruz         3.0    3.0     3.0   3.0   0.8                                  Enterococcus 373                                                                            7.5    7.5     7.5   3.0   3.0                                   0928/72      7.5    17.5    7.5   3.0   3.0                                  Escherichia coli                                                               10536        7.5    7.5     7.5   3.0   3.0                                   1574-1       7.5    7.5     3.0   3.0   3.0                                   777          3.0    7.5     3.0   3.0   3.0                                   4195         3.0    7.5     3.0   3.0   3.0                                   JR66         3.0    7.5     3.0   3.0   3.0                                  Klebsiella AD17                                                                             7.5    17.5    7.5   3.0   7.5                                   AD22         7.5    7.5     3.0   3.0   3.0                                   G3694        7.5    7.5     3.0   3.0   3.0                                   3020         7.5    7.5     3.0   3.0   3.0                                   121          7.5    17.5    3.0   3.0   7.5                                  Proteus mirabilis Hard                                                                      17.5   17.5    7.5   3.0   3.0                                   Peras        7.5    37.5    7.5   3.0   17.5                                 Proteus rettgeri                                                              mirabilis     17.5   17.5    7.5   7.5   17.5                                 Proteus vulgaris Napol                                                                      17.5   17.5    7.5   3.0   3.0                                  Proteus morganii Valz                                                                       17.5   17.5    7.5   3.0   17.5                                 Pseudomonas acruginosa                                                         8709         17.5   17.5    17.5  7.5   7.5                                   762          17.5   17.5    37.5  17.5  17.5                                  130          17.5   17.5    17.5  17.5  7.5                                   138          17.5   17.5    17.5  7.5   17.5                                  Trav. 1      17.5   17.5    17.5  7.5   17.5                                 Salmonella ent. 1                                                                           7.5    7.5     7.5   3.0   3.0                                   C.sub.2 Napc 3.0    7.5     3.0   3.0   3.0                                   B typhi      7.5    7.5     3.0   3.0   3.0                                   C.sub.1 Oao  7.5    7.5     3.0   3.0   3.0                                   C.sub.2 Cuban                                                                              7.5    7.5     3.0   3.0   3.0                                  ______________________________________                                    

                                      TABLE II                                    __________________________________________________________________________    In vitro activity of 2-aminoalkyl-3-phenylindoles of the formula I            against anaerobes in fluid Thioglycollate medium                              MIC (mcg/ml) after 48 and 72 hrs.                                                           Compound                                                                      A     B     C   D       E                                       Organism      48 72 48 72 48                                                                              72                                                                              48  72  48  72                                  __________________________________________________________________________     Bacteroides melaninogenicus                                                                7.5                                                                              7.5                                                                              7.5                                                                              17.5                                                                             7.5                                                                             7.5                                                                             3.0 3.0 3.0 3.0                                 Bacteroides fragilis                                                                        7.5                                                                              7.5                                                                              3.0                                                                              3.0                                                                              3.0                                                                             3.0                                                                             .75 .75 .75 .75                                 Bacteroides corrodens                                                                       7.5                                                                              7.5                                                                              7.5                                                                              7.5                                                                              3.0                                                                             3.0                                                                             .75 .75 .75 3.0                                 Eubacterium lentum                                                                          17.5                                                                             17.5                                                                             17.5                                                                             17.5                                                                             7.5                                                                             7.5                                                                             .75 .75 3.0 3.0                                 Clostridium novyi                                                                           7.5                                                                              7.5                                                                              7.5                                                                              7.5                                                                              3.0                                                                             3.0                                                                             .75 .75 .75 .75                                 Clostridium septicum                                                                        7.5                                                                              7.5                                                                              3.0                                                                              3.0                                                                              3.0                                                                             3.0                                                                             .75 .75 .75 .75                                 Clostridium histolyticum                                                                    7.5                                                                              7.5                                                                              7.5                                                                              7.5                                                                              3.0                                                                             3.0                                                                             .3  .3  .3  .3                                  Peptostreptococcus                                                                          3.0                                                                              3.0                                                                              3.0                                                                              3.0                                                                              3.0                                                                             3.0                                                                             .3  .3  .75 .75                                 __________________________________________________________________________

                  TABLE III                                                       ______________________________________                                        In vitro Anti-Candida Activity of 2-aminoalkyl-3-phenyl-                      indoles of the formula I. Dilutions in Sabouraud's Dextrose                   Agar                                                                          MIC (mcg/ml) after 48 hours                                                             Compound                                                            Candida albicans                                                                          A       B        C     D      E                                   ______________________________________                                        Burke       75      75       75    150    37.5                                Lusk        75      75       75    75     37.5                                Blunden     75      75       75    17.5   37.5                                Fix         75      75       75    75     37.5                                Collins     75      75       75    75     37.5                                Merkel      75      75       75    75     37.5                                29          75      75       37.5  75     37.5                                Wisconsin   75      150      75    150    37.5                                Sparks      75      150      75    150    37.5                                Bevan       75      150      75    150    37.5                                Newcomb       37.5  37.5     37.5  17.5   17.5                                Kennedy 1     37.5  75       37.5  17.5   37.5                                Pannell     75      75       75    75     17.5                                Atkisson    75      75       75    150    37.5                                1           75      150      75    150    37.5                                2           75      75       75    150    37.5                                3           75      150      75    75     37.5                                4           75      75       37.5  37.5   17.5                                5           75      75       75    75     17.5                                6           75      150      75    150    37.5                                8           75      150      75    150    37.5                                9           75      75       75    150    37.5                                Burnside    75      150      75    75     37.5                                Lehtman     75      150      75    150    37.5                                Fulcher     75      75       75    75     37.5                                Shurelli    75      150      75    150    37.5                                Frazier     75      150      75    150    37.5                                Boyer       75      150      75    150    37.5                                Sherod      75      150      75    150    37.5                                Bognay      75      75       75    75     37.5                                Johnson     75      150      75    150    37.5                                Gay         75      150      75    150    37.5                                Tyner       75      150      75    75     37.5                                Kenndy 2    75      150      75    150    37.5                                Bertrand    75      150      75    150    37.5                                Hons        75      150      75    150    37.5                                ______________________________________                                    

                                      TABLE IV                                    __________________________________________________________________________    In vitro Antifungal Activity of 2-amioalkyl-3-phenylindoles                   of the formula I against Dermetophytes and Aspergillus.                       Dilutions in Sabouraud's Dextrose Broth.                                      MIC (mcg/ml) after 96 hours                                                                 Compound                                                        Fungi         A    B    C    D    E                                           __________________________________________________________________________    Trichophyton ascoides                                                                       17.5 17.5 7.5  7.5  7.5                                          discoides    7.5  17.5 17.5 7.5  7.5                                          ferrugineum  7.5  37.5 17.5 17.5 7.5                                          gallinae     7.5  7.5  37.5 75.0 17.5                                         megninii     3.0  7.5  7.5  3.0  3.0                                          mentagrophytes A                                                                           7.5  7.5  7.5  7.5  3.0                                          mentagrophytes B                                                                           7.5  7.5  7.5  3.0  17.5                                         mentagrophytes C                                                                           7.5  7.5  3.0  3.0  17.5                                         mentagrophytes Young                                                                       7.5  7.5  7.5  7.5  3.0                                          mentagrophytes H377                                                                        7.5  17.5 17.5 7.5  7.5                                          rubrum Blehl 7.5  17.5 37.5 7.5  3.0                                          rubrum 3     7.5  17.5 17.5 3.0  3.0                                          rubrum Haggerty                                                                            7.5  17.5 7.5  7.5  3.0                                          rubrum 360   7.5  17.5 7.5  3.0  3.0                                          schoenleinii 17.5 17.5 17.5 7.5  3.0                                          schoenleinii 2                                                                             3.0  7.5  3.0  7.5  3.0                                          soudenense   17.5 17.5 17.5 3.0  7.5                                          tonsurans    17.5 200.0                                                                              7.5  17.5 7.5                                         Tricoderm sp. 7.5  7.5  7.5  7.5  3.0                                         Microsporum nanum                                                                           3.0  17.5 3.0  3.0  3.0                                         Microsporum distortum                                                                       3.0  17.5 7.5  3.0  3.0                                         Epidermophyton floccosum                                                                    3.0  17.5 17.5 7.5  3.0                                         Aspergillus sp. No. 3                                                                       17.5 37.5 17.5 17.5 17.5                                        Aspergillus niger 6275                                                                      17.5 37.5 17.5 17.5 17.5                                        __________________________________________________________________________

                  TABLE V                                                         ______________________________________                                        Acute Toxicity of 2-aminoalkyl-3-phenylindoles of the formula I               in mice.                                                                      Acute LD.sub.50 (mg/kg)                                                       Compound   I.P.     S.C.     Oral                                             ______________________________________                                        A          250      850            >1000                                      B          225      900      about 1000                                       C          100      600            >1000                                      D          175      750      about 950                                        E           85      540      about 700                                        ______________________________________                                    

                  TABLE VI                                                        ______________________________________                                        Effect of some 2-aminoalkyl-3-phenylindoles of the formula II                 against topical T. mentagrophytes infections in guinea pigs.                                   Average No. of                                                                Days to become                                                                             Average Sum of                                  Com-   Concen-    negative    lesion scores                                   pound  tration   Cultures Lesion                                                                              Days 1-5                                                                             Days 1-10                              ______________________________________                                        A      1%        6.8      9.0   11.8   16.0                                   "      4%        6.0      8.8   11.2   14.8                                   B      1%        9.6      14.2  11.6   20.0                                   "      4%        5.0      7.2   9.2    10.0                                   C      1%        12.4     14.4  13.0   25.0                                   "      4%        5.6      7.4   10.0   11.8                                   D      1%        >22      >22   11.6   29.2                                   "      4%        6.2      8.8   11.8   14.2                                   E      1%        >22      >22   12.6   34.2                                   "      4%        6.6      9.2   11.6   15.2                                   Untreated controls                                                                          >22      >22     14.8   38.2                                    ______________________________________                                    

                  TABLE VII                                                       ______________________________________                                        In vitro Anti-Trichomonal Activity of some 2-aminoalkyl-3-                    phenyl-indenes of formula II.                                                          Minimal Level (mcg/ml) to Produce                                    Compound   90% Suppression                                                                              50% Suppression                                     ______________________________________                                        A          40             24                                                  B          75             32                                                  C          38             30                                                  D          38             20                                                  E          28             14                                                  ______________________________________                                    

The invention, therefore, provides compositions containing, as an activeingredient, a 2-aminoalkyl-3-phenyl-heteroindene of the formula Ihereinbefore defined or an acid addition salt thereof, in associationwith a suitable carrier, excipient or diluent. In its function as activeingredient, the compound of the formula I or salt thereof may be used topreserve the carrier from microbial contamination; for example, thecarrier may be cutting or other oil, paper, leather, photographicemulsion, canvas or rope. If the salt is non-toxic, the carrier may alsobe a food-stuff, food-additive or food-supplement, or a medicinal orcosmetic preparation. Such medicinal or cosmetic preparations mayconveniently be in fluid form, e.g., lotions, creams, ointments,solutions, suspensions or aerosol preparations.

When used as preservatives, the compounds of the formula I or theirsalts are preferably incorporated into the composition to be preservedin an amount of 0.05 to 1% by weight, especially 0.1 to 0.5% by weight.

The compounds of formula I and their non-toxic acid addition salts canthemselves be used in medicine as anti-microbial agents, and thus may beformulated as pharmaceutical compositions containing at least one saidcompound or salt together with a pharmaceutical carrier or excipient.Such a composition may, for example, be in the form of shaped products,in particular dosage units, such as pills, tablets, capsules, dragees,lozenges or suppositories (especially vaginal suppositories).Alternatively, such compositions may be adapted for injection andtherefore have as carrier a sterile, pyrogen-free injectable liquid.Injectable compositions will normally be in the form of dosage units;the various dosage units mentioned conveniently contain from 2 to 100mg., preferably from 5 to 50 mg., of a compound of formula I ornon-toxic acid addition salt thereof.

Compositions for oral administration, other than dosage units mentionedabove, may be exemplified by powders, granulates, solutions,suspensions, elixirs, or aerosols. Compositions for topical applicationmay be exemplified by ointments, creams, lotions, solutions,suspensions, aerosols, gels, shampoos, soaps or dusting powders. Thecompositions may be adapted in particular as ophthalmic, otic and nasalpreparations. Such compositions will normally be based upon standardcarriers such as those selected from pharmaceutically acceptablevegetable oils, pharmaceutically acceptable polyalkylene glycols,isopropanol, gelatin, benzyl alcohol, gums, glycerol, petrolatum,preservatives starch, sugars such as lactose, talc, magnesium stearate,aerosol propellants such as chlorofluoroalkanes, and coloring,flavoring, sweetening, thickening, suspending, dispersing, emulsifying,wetting, stabilising and buffering agents.

The composition may also be in the form of an animal feed-stock,feed-additive or feed-supplement.

Compositions in which the active ingredient is a compound of the formulaI or non-toxic acid addition salt thereof preferably contain from 0.5 to10% thereof.

A suitable parenteral dosage range of the compounds of the formula I andnon-toxic acid addition salts thereof is about 2 to 10 mg./kg./day. Thecompounds of formula I and their non-toxic acid addition salts may beformulated into dosage forms as the sole active ingredient or used inassociation with other ingredients to extend the therapeutic spectrum.

The following formulations exemplify pharmaceutical compositionscontaining 2-aminoalkyl-hetero-indenes of this invention; the activeingredient illustrated may, of course, be replaced with another compoundof formula I or non-toxic acid addition salt thereof, e.g., compound Dor E or especially B from the foregoing tests.

    ______________________________________                                        Formulation 1                                                                 Topical Cream         Per kg.                                                 ______________________________________                                        2-Aminomethyl-5-chloro-3-                                                     phenylindole          10 g. - 100 g.                                          Ethoxylated Cetyl/Stearyl Alcohol                                                                   20 g.                                                   Cetyl Alcohol         35 g.                                                   Stearyl Alcohol       35 g.                                                   Petrolatum            200 g.                                                  Mineral Oil           50 g.                                                   Buffers, Sufficient   --                                                      Preservatives, Sufficient                                                                           --                                                      Purified Water to make                                                                              1.0 kg.                                                 ______________________________________                                    

Add the cetyl alcohol, stearyl alcohol, ethoxylated cetyl/stearylalcohol, petrolatum and mineral oil to a suitable mixing vessel. Heat to80° C. to melt. Mix. Add the preservatives, buffers and2-aminomethyl-5-chloro-3-phenylindole in approximately 95% of thepurified water heated to 80° C. in a suitable mixing vessel. Mix. Addthe melted wax phase to the aqueous phase and mix while cooling to about40° C. Add sufficient purified water to make 1 kg. Mix until cool.

    ______________________________________                                        Formulation 2                                                                 Topical Ointment      Per kg.                                                 ______________________________________                                        2-Aminomethyl-5-chloro-3-                                                     (2-fluorophenyl)indole                                                                              10 g. - 100 g.                                          White Petrolatum, to make                                                                           1.0 kg.                                                 ______________________________________                                    

Melt and heat the petrolatum to 50° C. in a suitable mixing vessel.Remove a portion of the melted petrolatum and make therewith a slurry ofthe 2-aminomethyl-5-chloro-3-(2-fluorophenyl)indole. Pass the slurrythrough a suitable colloid mill and mill until a uniform dispersion isobtained. Add the milled slurry to the remainder of the meltedpetrolatum and mix until cool.

    ______________________________________                                        Formulation 3                                                                 Otic Suspension        mg/ml                                                  ______________________________________                                        2-aminomethyl-5-chloro-3-                                                     (2-fluorophenyl)indole 5 - 10                                                 Cetylpyridinium Chloride, NF                                                                         0.20                                                   Glyceryl Triacetate    880.0                                                  Polyethylene Glycol 200 q.s. ad                                                                      1.0 ml                                                 ______________________________________                                    

Melt and heat the petrolatum to 50° C. in a suitable mixing vessel.Remove a portion of the melted petrolatum and make a slurry of the2-aminomethyl-5-chloro-3-(2-fluorophenyl)indole. Pass the slurry througha suitable colloid mill and mill until a uniform dispersion is obtained.Add the milled slurry to the remainder of the melted petrolatum and mixuntil cool.

    ______________________________________                                        Formulation 4                                                                                        mg/      mg/                                           Vaginal Tablets        tablet   tablet                                        ______________________________________                                        2-Aminomethyl-5-chloro-3-                                                     (2-fluorophenyl)indole 10.0      5.0                                          Lactose Hydrous, Impalpable powder USP                                                               772.0    777.0                                         Sodium Lauryl Sulfate  20.0     20.0                                          Polyvinylpyrrolidone   40.0     40.0                                          Corn Starch, Food Grade                                                                              150.0    150.0                                         Magnesium Stearate      8.0      8.0                                                                 1000 mg  1000 mg                                       ______________________________________                                    

    ______________________________________                                        Formulation 5                                                                 Intramuscular or Subcutaneous Oil                                             Injection              mg/ml                                                  ______________________________________                                        2-aminomethyl-5-chloro-3-                                                     (2-fluorophenyl)indole 10 - 50                                                Aluminium Monostearate, USP                                                                          20.0                                                   Sesame Oil, Heated Treated, USP qs ad                                                                1.0 ml                                                 ______________________________________                                    

Formulations 6 to 8 illustrate compositions preserved with a2-aminomethyl-3-phenylindole of the formula I:

    ______________________________________                                        Formulation 6                                                                 Lotion                 mg/ml                                                  ______________________________________                                        Betamethasone Valerate 1.22                                                   2-aminomethyl-5-chloro-3-                                                     (2-fluorophenyl) indole HCl                                                                          1.00                                                   Mineral Oil, USP       19.50                                                  Diethylene Glycol Monostearate S.E.                                                                  6.50                                                   Cetostearyl Alcohol    6.50                                                   Lanbritol Wax          9.30                                                   Glycerin, USP          50.00                                                  Isopropanol            65.00                                                  Citric Acid            0.08                                                   Purified Water, USP, to make                                                                         1.00 ml                                                ______________________________________                                    

    ______________________________________                                        Formulation 7                                                                 Intramuscular or Intravenous Solution                                                                mg/ml                                                  ______________________________________                                        Gentamicin (charged as sulfate)                                                                      40.0                                                   Sodium Bisulfite, USP  3.2                                                    Disodium Edetate, USP  0.1                                                    2-aminomethyl-5-chloro-3-(2-fluorophenyl)-                                    indole (charged as the hydrochloride salt)                                                           1-3                                                    Water for Injection qs ad                                                                            1.0 ml                                                 ______________________________________                                    

    ______________________________________                                        Formulation 8                                                                 Aerosol Concentrate    mg/g                                                   ______________________________________                                        Megalomicin A Phosphate                                                                              20.0                                                   2-aminomethyl-5-chloro-3-                                                     (2-fluorophenyl) indole                                                                               1.0                                                   Liquid Absorption Base 90.0                                                   Stearic Acid           25.0                                                   Glyceryl Monostearate  25.0                                                   Isopropyl Myristate    50.0                                                   Glycerol, USP          100.0                                                  Alcohol SD 40          80.0                                                   Triethanolamine        10.0                                                   Purified Water, USP, to make                                                                          1.0 g                                                 ______________________________________                                    

This composition is packaged into a aerosol container with standardpolyfluoroalkane propellant mixtures.

    ______________________________________                                        Formulation 9                                                                 Topical Cream           Per kg.                                               ______________________________________                                        2-Aminomethyl-5-bromo-3- phenylbenzob]                                        thiophene hydrochloride 10 g. - 100 g.                                        Stearic acid            60 g.                                                 Propylene Glycol Monostearate                                                                         100 g.                                                Isopropyl Myristate     80 g.                                                 Polyoxyethylene (20) Sorbitan                                                 Monopalmitate           60 g.                                                 Sorbitan Solution       20 g.                                                 Buffers, Sufficient     --                                                    Preservatives, Sufficient                                                                             --                                                    Purified Water to make  1.0 kg.                                               ______________________________________                                    

Add the stearic acid, propylene glycol monostearate, isopropyl myristateand polyoxyethylene (20) sorbitan monopalmitate to a suitable mixingvessel. Heat to 80° C. to melt. Mix.

It is further contemplated that the compounds of the present inventionalso have herbicidal, insecticidal and other biocidal activity. Thecompounds may have selective and/or broad base activity depending uponthe specific compound within formula I and the specific use for which itis applied.

When used as a herbicide, the compounds may be applied to a stand ofcrops and weeds in the post-emergence treatment and to the ground in thepre-plant or pre-emergence treatment in a number of ways, well known tothe art. The water-soluble compounds, such as the hydrochloride salts,may be sprayed simply as alcoholic/aqueous solutions. The compounds canbe deposited as dusts containing a powdered carrier such as talc,attaclay, etc. The compounds having limited water solubility, such asthe amine base itself, can be applied as emulsions, the same beingformulated as is well known in the art, with commercially availablesurface-active agents. Among the surface-active agents utilizable arethe sulfonated vegetable oils, sodium lauryl sulfate, Tween No. 20 (apolyalkylene ether alcohol), carbowax (polyethylene glycols of M.W. 1500or more), Atlas G-2122 (polyoxyethylene glycol monolaurate), etc.Penetrants, sequestrants, mineral oils and cosolvents may also beincluded in the formulations. An example of a suitable formulation isgiven herein below:

Formulation 10 2-amino methyl-5-bromo-3-phenybenzo[b]thiophenehydrochloride ethanol.

This ethanol water formulation, when applied on the basis of 4-8 lbs. ofamine base, may be used on a pre-emergence basis against weeds, such asbarnyard grass, crabgrass and chickweed.

The compounds may be used as a dusting powder or spray as abovedescribed against insects such as ringworm and brown planthopper.

I claim:
 1. A method for preserving a compound from microbialcontamination, which comprises incorporating, in a composition in whichpreservation is desired, about 0.1 to 0.5% by weight of a compound ofthe formula: ##STR4## wherein n is 0 or 1; R₂ is hydrogen, or loweramino alkylene having 1 to 3 carbon atoms; R₁ is carbonyl or loweralkylene having 1 to 3 carbon atoms with the provisio that when R₂ ishydrogen, R₁ is lower alkylene; T is S; M is hydrogen, halogen, methylor trifluoromethyl; X is halogen, nitro, methoxy or trifluoromethyl; Yis hydrogen, halogen or methyl with the provisio that when X is nitro, Yis halogen; Z is hydrogen, halogen, nitro or methyl; and the non-toxicacid addition salts thereof.